1. Field of the Invention
The present invention relates to a Corynebacterium glutamicum pyruvate carboxylase protein and to polynucleotides encoding this protein.
2. Background Information
Pyruvate carboxylate is an important anaplerotic enzyme replenishing oxaloacetate consumed for biosynthesis during growth, or lysine and glutamic acid production in industrial fermentations.
The two-step reaction mechanism catalyzed by pyruvate carboxylase is shown below: ##EQU1##
In reaction (1) the ATP-dependent biotin carboxylase domain carboxylates a biotin prosthetic group linked to a specific lysine residue in the biotin-carboxyl-carrier protein(BCCP)domain. Acetyl-coenzyme A activates reaction (1) by increasing the rate of bicarbonate-dependent ATP cleavage. In reaction (2), the BCCP domain donates the CO.sub.2 to pyruvate in a reaction catalyzed by the transcarboxylase domain (Attwood, P. V., Int. J. Biochem. Cell. Biol. 27:231-249 (1995)).
Pruvate carboxylase genes have been cloned and sequenced from: Rhizobium etli (Dunn, F. F., et al., J. Bacteriol. 178:5960-5970 (1996)), Bacillus stearothermophilus (Kondo, H., et al., Gene 191:47-50 (1997), Bacillus subtillis (Genbank accession no. Z97025), Mycobacterium tuberculosis (Genbank accession no. Z83018), and Methanobacterium thermoautotrophicum (Mukhopadhyay, B., J. Biol. Chem. 273:5155-5166 (1998). Pyruvate carboxylase activity has been measured previously in Brevibacterium lactofermentum (Tosaka, O., et al., Agric. Biol. Chem. 43:1513-1519 (1979)) and Corynebacterium glutamicum (Peters-Wendisch, P. G., et al., Microbiology 143:1095-1103 (1997).
Previous research has indicated that the yield and productivity of the aspartate family of amino acids depends critically on the carbon flux through anaplerotic pathways (Vallino, J. J., & Stephanopoulos, G., Biotechnol. Bioeng. 41:633-646 (1993)). On the basis of the metabolite balances, it can be shown that the rate of lysine production is less than or equal to the rate of oxaloacetate synthesis via the anaplerotic pathways.